PapQuin-50 a quinoa grain-based bacterial growth medium nitrogen source

ABSTRACT

Commercially available Quinoa grain powder is subjected to proteolytic digestion using papain enzyme. Derived hydrolysate is ultrafiltered using two sequential ultrafiltration steps of 50 KDa and 5 KDa membranes to remove high and low molecular impurities respectively. Concentrated filtrate is 0.2 μ filter-sterilized and used as a component growth medium for fastidious microorganisms  Neisseria meningitidis  and  Streptococcus pneumoniae . The product is tested for its ability to be used as meat free nitrogen and carbon source for the fastidious bacterial growth and found to be working well.

BACKGROUND OF THE INVENTION 1. Field of Invention

The current invention is related to the fields of Medical Microbiology,Industrial Microbiology, Biotechnology, and Bacterial VaccineManufacturing.

2. Description of the Prior Art

Purified extracts from meat [example: Beef extract, peptones] and animalproducts such as milk derived casein enzyme hydrolysates [example:Casein hydrolysate], yeast cell extracts [example: Yeast extract] andplant origin seed extracts [example: Hy-Soy] have been used as nitrogenand/or carbon sources in microbial growth media formulations Peptonesare derived from meat or milk and Tryptones are derived from only milk.A mixture of animal origin pancreatic digestive enzymes amylase, lipaseand protease called ‘pancreatin’ is used for meat or milk protein caseinhydrolysis to produce peptones and tryptones respectively. Differentvarieties of Soya bean protein-based enzyme hydrolysates Peptone Hy-Soy,Peptone N—Z-Soy and Soy protein acid hydrolysates are already existingas commercial products. Water soluble, acid hydrolysates, enzymatichydrolysates of above sources are often proven their promising role inbacterial growth media. Commercial bacterial fermentations are widelytaking place in pharmaceutical industries not only to produce differentcomponents including antigens from cultured microbes but also to expressrecombinant proteins in them. Commercial fermentation media formulationsare demanding the importance of plant-based components because of animalcomponent related health issues for example, bovine spongiformencephalopathy in cattle. A search for highly nutritious plant-basedproducts is currently growing more than ever. Growth of fastidiousmicrobes is dependent on specific nutrient growth factors of the medium.Yeast (extract) is generally recognized as safe (GRAS) by USFDA andwidely used currently in bio-pharma fermentation media. However, thereare many issues associated with yeast extract of lot-to-lot variationthat causes batch to batch fermentation growth and yield variations.Yeast extracts are produced by culturing source organism ex.Saccharomyces cerevisiae using molasses in fermentation media. Grownyeast cells are lysed in multiple ways including, mechanical,non-mechanical, invasive, or non-invasive ways. These processvariabilities can cause variations on the composition of yeast extractproduct. Plant derived growth supplements can pose lesser processingvariability and hence can be more consistent in their batch fermentationperformance. Quinoa is common name for Chenopodium quinoa a smallflowering plant of family Amaranthaceae. Quinoa seed (grain) apseudo-cereal is edible that comes in various colors, mainly white,yellow, red, and black. More than 120 known varieties of quinoa areavailable. Quinoa grain is gluten-free high protein source containingall essential amino acids, magnesium, B-vitamins, iron, potassium,calcium, manganese, phosphate and vitamin E. The plant has beencultivated for more than 5000 years and indigenous to the Andean regionof South America. It had been introduced in Europe, North America, Asia,and Africa recently. Quinoa fluor contains nearly 16% protein whilewhite wheat flour contains a 10% protein. Quinoa seed is a naturallygluten-free and fluor is used widely in human food consumption ashealthy protein source for celiac patients. Quinoa beer was developed byde Meo et al (2011).

Quinoa flour slurry was recently used as medium component in lactic acidbacterial fermentation (Dallagnol A M et al. 2013).

REFERENCES

-   De Meo B, Freeman G, Marconi O, Booer C, Perretti G, Fantozzi    P, (2011) Behaviour of malted cereals and pseudo-cereals for    gluten-free beer production. J Inst Brew 117:541-546.-   Dallagnol A M, Pescuma M, Valdez G F D, and Rollan G (2013)    Fermentation of quinoa and wheat slurries by Lactobacillus plantarum    CRL 778: proteolytic activity. Appl Microbiol Biotechnol    97(7):3129-3140.

BRIEF SUMMARY OF THE INVENTION

Plant based microbial growth media components are gradually replacinganimal derived components. Quinoa seed grain powder (flour) is rich inproteins, carbohydrates, vitamins, and minerals. This invention,describes a method of preparing plant based quinoa flour mediumcomponent, digestion by using plant based enzyme papain, extraction andpurification procedure. Commercially available quinoa powder [Bob's RedMill Organic Quinoa Flour] was subjected to papain enzymatic hydrolysis.A 50 KDa molecular weight cut-off [MWCO] cassette membraneultrafiltered, derived hydrolysate permeate named herein as PapQuin-50was tested for its suitability on fastidious bacterial growth. w Papainalso known as papaya proteinase, is a cysteine protease that cleavespeptide bonds of basic amino acids, leucine, or glycine. Papain pHoptimum is 6.0-7.0 and the temperature optimum is 60-70° C. It alsohydrolyzes esters and amides. Papain digests most protein substratesmore extensively than pancreatic proteases.

Source of the quinoa flour must be identified/secured for batchconsistency of the product. In this submission a commercially availableorganic quinoa powder is used.

Derived hydrolysate is ultrafiltered using 50 KDa MWCO membrane cassetteto remove high-molecular impurities including the saponins and permeatewas collected. The permeate is then purified by 5 KDa MWCO membraneultrafiltration to collect retentate. Concentrated retentate is 0.2μfilter-sterilized and used as growth medium for fastidiousmicroorganisms Neisseria meningitidis and Streptococcus pneumoniae. Theproduct is tested for its ability to be used as meat free nitrogen andcarbon source for the fastidious bacterial growth and found to beworking well.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Process used in the current invention:

-   -   Water solubilized quinoa powder is prepared at a concentration        of 50 mg/ml by constant mixing at 37° C. for 2 to 3 h. The        solution is then filtered through 0.8 μm filter to separate        undissolved portion.    -   Filtered Quinoa solution is then pH adjusted to 7.0 and treated        with papain [Thermo Scientific Cat #AC416761000] enzyme at a        final concentration of 50 μg/ml at 65° C. for 6 h with constant        shaking in a shaker incubator at 150 RPM.    -   Centrifuged the hydrolysate at 8000 g at 4° C. for 30 minutes to        separate any insoluble sediment.    -   Transferred the supernatant to ultrafiltration step in a        Viva-flow [Sartorius item #VF05P3] 50 KDa MWCO PES membrane        cassette on a laboratory scale cross flow system.    -   Collected the flow-though (Permeate) at a twice starting        concentration (100 mg/ml) based on collected flow-through        volume.    -   The Permeate is further purified with Viva-flow [Sartorius item        #VFO5P1] 5 KDa MWCO membrane cassette ultrafiltration system for        5 diafiltration-volumes of purified water to remove small        molecular contamination to collect the retentate.    -   0.2 micron Filter sterilized product PapQuin-50 was used as a        medium component substituting yeast extract in Frantz medium of        composition given below table 1.    -   Tested the medium agar plates and liquid growth for fastidious        organism Neisseria meningitidis serogroups A, C, W-135 and Y and        Streptococcus pneumoniae serotypes 7F, 9V, 12F and 23F. 15 g/L        Bacto agar is used for making agar plates.    -   Results showed that Papquin-50 can replace yeast extract in        Frantz medium to support the agar plate and liquid medium growth        of Neisseria meningitidis and Streptococcus pneumoniae strains        used.

TABLE 1 Medium composition: Frantz medium replacing Yeast extract withPapQuin-50 *Frantz media Current-work Component g/L medium g/LL-glutamic acid 1.60 1.60 L-Cysteine · HCl 0.02 0.02 NaCl 6.00 6.00Na₂HPO₄, 12H₂O 6.24 6.24 KCl 0.09 0.09 NH₄Cl 1.25 1.29 MgSO₄, 7H₂O 1.231.23 Glucose 5.00 5.00 PapQuin-50 — 2.00 Yeast Extract 2.00 —

-   -   Frantz I. D. Jr Growth Requirements of the meningococcus. J.        Bact (1942). 43:757-761, 1        Results:    -   Papquin-50 used to replace the yeast extract in Frantz medium        worked very well for both Neisseria and Streptococcus strains        used.    -   Papquin-50 is shown to be working for agar plate growth and        liquid medium growth during the invention.

CONCLUSIONS

Plant origin papain enzyme digested quinoa flour nitrogen source named‘PapQuin-50’ preparation is described in this patent application.Commercially available Quinoa grain powder used in this claim isdigested with commercially available plant origin enzyme papain. A MWCO50 KDa and 5 KDa membrane diafiltration steps are used to purify thisnitrogen source to make consistent preparation. Growth promotingactivity of the PapQuin-50 medium replacing yeast extract in a standardmedium supporting fastidious Gram negative (meningococci) and Grampositive (Pneumococci) bacteria has been presented. This nutritiousmedium supplement Papquin-50 may support the growth of other bacterialspecies as well.

The invention claimed is:
 1. A growth medium composition for growingfastidious bacteria Neisseria meningitidis and Streptococcus pneumoniae,the growth medium comprising an ultrafiltration fraction of a quinoagrain papain hydrolysate in the molecular weight range of 5 kDa to 50kDa, wherein said ultrafiltration fraction is supplemented at aconcentration of 2 g/L to a bacterial growth medium lacking yeastextract to form said growth medium composition that supports the growthof the fastidious bacteria Neisseria meningitidis and Streptococcuspneumoniae.
 2. A method for preparing a growth medium compositioncomprising quinoa grain papain hydrolysate comprising an ultrafiltrationfraction in the molecular weight range of 5 kDa to 50 kDa for growingfastidious bacteria Neisseria meningitidis and Streptococcus pneumoniae,the method comprising the steps of: i) performing enzymatic hydrolysisof an aqueous solution of quinoa grain powder by papain to obtain aquinoa grain papain hydrolysate, ii) performing purification of thepapain hydrolysate using sequential ultrafiltration steps with membraneshaving 50 kDa molecular weight cutoff (MWCO) followed by 5 kDa MWCOmembrane filtration to obtain a retentate, iii) filter sterilizing saidretentate to obtain the quinoa grain papain hydrolysate comprising theultrafiltration fraction in the molecular weight range of 5 kDa to 50kDa, and iv) adding the sterilized retentate to a bacterial growthmedium that does not comprise yeast extract, at a concentration of 2g/L, to prepare the growth medium composition comprising the quinoagrain papain hydrolysate.